Isoflavone-beta-D-glucan produced by Agaricus blazei in the submerged liquid culture and method of producing same

ABSTRACT

The present invention relates to low and medium molecular weight isoflavone-β-D-glucan produced by submerged liquid culture of  Agaricus blazei,  a method of producing the isoflavone-β-D-glucan using autolysis enzyme of  Agaricus blazei  mycelia, and use of the isoflavone-β-D-glucan for anti-cancer and immunoenhancing effect.

CROSS REFERENCE TO RELATED APPLICATION

This application is a divisional application of U.S. patent applicationSer. No. 10/890,537 filed Jul. 12, 2004, which claimed the priority ofKorean Patent Application No. 2003-67439, filed Sep. 29, 2003, in theKorean Intellectual Property Office, the contents of which areincorporated by reference herein in their entirely.

TECHNICAL FIELD

The present invention relates to low and medium molecular weightisoflavone-β-D-glucan produced by submerged liquid culture of Agaricusblazei, a method of producing the isoflavone-β-D-glucan using autolysisenzyme of Agaricus blazei mycelia, and use of the isoflavone-β-D-glucanfor anti-cancer and immunoenhancing effect.

BACKGROUND ART

Cancer holds the most fatal disease in the statistics of mortality, sothat anti-cancer agents have been a matter of continued concern.Conventional anti-cancer agents have much toxicity to affect normalcells since they are lack of specificity to cancer cells, which resultsin a variety adverse effects such as depilation, lowering of immunityand liver function, etc.

Considering the above, various researches have been carried out toobtain natural anti-cancer agents from all sorts of food having no orlittle adverse effect. As a result, it is revealed that β-D-glucanobtained from mushroom mycelia and isoflavone contained in soybean, etc.have anti-cancer effect.

β-D-glucan is a main bioactive component in mushroom. It is found thatthe anti-cancer activity of mushroom derives from the activation ofimmunocyte by β-D-glucan to delay the progress of cancer and to preventthe transition of cancer. Accordingly, β-D-glucan may be used along withcancer treatment to improve the effect.

β-D-glucan, extract from mycelium culture of mushroom, is produced on acommercial scale in some advanced countries. For example, AHCC, which isimported from Japan, is produced by mixing extracts from myceliumcultures of seven mushrooms and is distributed through sales network forhospital to cancer patients. Further, arabinoxylan, which is extractfrom Lentinus edodes mycelia, is distributed in the US. In Korea,extract of Phellinus linteus mycelia, which is produced on a commercialscale by the trade name of Mesima-EX (HAN KOOK SIN YAK Corp, Korea), ison the clinical test.

The extracts of mushroom mycelium culture commercialized by this timehave not been distributed on a large scale since the extraction processcosts very high. The amount of extract of mushroom mycelium culture,that is, the amount of polysaccharides depends on the cultivation periodof mushroom and the growth rate of mycelia. In general, the cultivationperiod of Lentinus edodes, Phellinus linteus, Ganoderma lucidum, etc. islong and the growth of mycelia is slow, which limits the amount ofextracts.

The polysaccharides extracted from mycelium culture require to behydrolyzed to medium or low molecular weight polysaccharides in order tobe absorbed easily in the body. However, the enzyme for thehydrolyzation is hard to obtain, which makes the cost high. Further, themethod of separating and purifying the medium or low molecular weightpolysaccharides produced from the hydrolyzation has technicaldifficulty, so that the yield is very low.

Isoflavone, contained mainly in soybean, exists as aglycon form (e.g.,daidzein, genistein or glycitein), as glycoside form thereof (e.g.,daidzin, genistin or glycitin), as acetylglycoside thereof (e.g.,acetylglucose), or as malonylglycoside thereof (e.g., malonylglucose).It is found that isoflavone has anti-mutagenic and anti-cancer effect.The anti-cancer effect of isoflavone is mainly caused by genistein inthe aglycon form, and the glycoside form is not known to haveanti-cancer effect.

Considering the above, efforts have continued for the development of ananti-cancer agent with immunoenhancing effect, without affectingadversely on normal cell, and a method of preparing the agent in greatquantities with low cost.

DISCLOSURE OF THE INVENTION

It is an object of the present invention to provide a glycoside ofisoflavone and β-D-glucan having anti-cancer effect and immunoenhancingeffect, and a method of producing the glycoside from mushroom myceliumculture.

In accordance with one aspect of the present invention, it is provided amethod of producing isoflavone-β-D-glucan, which comprises the steps of:

culturing Agaricus blazei mycelia in a liquid medium containingisoflavone to produce a high molecular weight isoflavone-β-D-glucan;

separating the high molecular weight isoflavone-β-D-glucan from theliquid culture of Agaricus blazei mycelia;

separating an autolysis enzyme from a separate liquid culture ofAgaricus blazei mycelia;

adding the autolysis enzyme to the high molecular weightisoflavone-β-D-glucan to produce a low and medium molecular weightisoflavone-β-D-glucan; and

separating and purifying the low and medium molecular weightisoflavone-β-D-glucan.

Preferably, the step of separating the high molecular weightisoflavone-β-D-glucan from the liquid culture of Agaricus blazei myceliacomprises:

extracting the liquid culture of Agaricus blazei mycelia with boilingwater and concentrating the extract;

adding ethanol to the concentrated extract to make precipitation; and

separating the precipitate.

In the above step of separating the high molecular weightisoflavone-β-D-glucan from the liquid culture of Agaricus blazeimycelia, ethanol is preferably added in the concentration of 80%.

Preferably, the step of separating an autolysis enzyme from a separateliquid culture of Agaricus blazei mycelia comprises:

filtrating the liquid culture of Agaricus blazei mycelia under reducedpressure;

adding trichloroacetic acid to the filtered liquid culture of Agaricusblazei mycelia to make precipitation; and

separating the precipitate.

In the above step of producing a low and medium molecular weightisoflavone-β-D-glucan, the autolysis enzyme is preferably added to thehigh molecular weight isoflavone-β-D-glucan at pH 4.5-5.5.

In accordance with another aspect of the present invention, it isprovided a method of producing isoflavone-β-D-glucan, which comprisesthe steps of:

culturing Agaricus blazei mycelia in a liquid medium containingisoflavone to produce a high molecular weight isoflavone-β-D-glucan;

activating an autolysis enzyme in the liquid culture of Agaricus blazeimycelia to produce a low and medium molecular weightisoflavone-β-D-glucan; and

separating and purifying the low and medium molecular weightisoflavone-β-D-glucan.

Preferably, the step of activating an autolysis enzyme from the liquidculture of Agaricus blazei mycelia is carried out by adjusting the pH ofculture medium to pH 4.5-5.5 for 1-3 hours.

In accordance with still another aspect of the present invention, it isprovided a low and medium molecular weight isoflavone-β-D-glucanproduced by the above-mentioned method.

The low and medium molecular weight isoflavone-β-D-glucan produced froma liquid culture of Agaricus blazei mycelia is used as anti-cancer andimmunoenhancing agent.

Generally, polysaccharides having anti-cancer activity have beenobtained from Basidiomycetes by extracting fruit body or myceliumcultured in solid medium or liquid medium. In solid culture, a longperiod of time is required for culturing and the process of extractingthe anti-cancer polysaccharides is difficult. Further, the amount ofpolysaccharides extracted from fruit body is very low, and therefore, itis difficult to produce them on a large scale. On the other hand, inliquid culture, a short period of time is required, and it is possibleto culture in a settled condition, which enables mycelia including aregular content of polysaccharides to be obtained on a large scale atlow cost.

The present invention is characterized in using Agaricus blazei myceliaas the mushroom mycelia cultured in liquid media containing isoflavoneto produce low and medium molecular weight isoflavone-β-D-glucan.

According to the present invention, low and medium molecular weightisoflavone-β-D-glucan is prepared from high molecular weightisoflavone-β-D-glucan extracted from liquid culture of Agaricus blazeimycelia, in order to enhance internal absorption thereof. In the presentinvention, autolysis enzyme secreted by Agaricus blazei mycelia is usedto digest the high molecular weight isoflavone-β-D-glucan at an optimumcondition for the activity of the enzyme. Especially, Agaricus blazeimycelium is selected for its highest activity of autolysis enzyme.

Low and medium molecular weight isoflavone-β-D-glucan produced accordingto the present invention may be used for anti-cancer and immunoenhancingeffect. The isoflavone-β-D-glucan of the present invention is veryeffective in killing cancer cells and shows no toxic effect on normalcells. It is found that isoflavone in free form has anti-cancer effect.However, the anti-cancer effect of isoflavone-β-D-glucan as glycosidehas never been found before the present invention.

Low and medium molecular weight isoflavone-β-D-glucan produced accordingto the present invention is characterized to have anti-cancer effect aswell as immunoenhancing effect. The anti-cancer effect of the low andmedium molecular weight isoflavone-β-D-glucan is remarkably increasedwith immunoenhancing effect.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects, features and other advantages of thepresent invention will be more clearly understood from the followingdetailed description taken in conjunction with the accompanyingdrawings, in which:

FIG. 1 shows a process of preparing a low and medium molecular weightisoflavone-β-D-glucan according to the present invention;

FIG. 2 is HPLC chromatograms of the extract of liquid culture ofAgaricus blazei mycelia treated with various concentrations of ethanolsolution.

FIG. 3 shows fractionation of the precipitate of the liquid culture ofAgaricus blazei mycelia by DEAE column chromatography;

FIG. 4 is UV spectrum of DEAE column fraction #2 (tube #8) at 225-445nm;

FIG. 5 shows SDS-PAGE pattern of tubes #8 and #14;

FIG. 6 shows TLC pattern of 80 EP reacted with autolysis enzyme;

FIGS. 7 a and 7 b are HPLC chromatograms of each fraction containingautolysis enzyme separated from HPLC (TSK column) before and after beingtreated with 80 EP;

FIG. 8 is HPLC chromatograms of the products from 80 EP treated withtube #8, which are detected with UV detector and RI detector;

FIG. 9 shows TLC pattern of soybean powder and its acid hydrolyzate;

FIG. 10 is UV/VIS spectra of autolysis enzyme-treated 80 EP;

FIGS. 11 a, 11 b and 11 c are IR spectra of TLC bands of FIG. 9;

FIG. 12 shows another process of preparing a low, and medium molecularweight isoflavone-β-D-glucan according to the present invention;

FIG. 13 is a graph showing molecular weight of low and medium molecularweight isoflavone-β-D-glucan according to the present inventioncalculated by comparing with dextran standard and displayed in logscale;

FIG. 14 is IR spectrum of low and medium molecular weightisoflavone-β-D-glucan according to the present invention;

FIG. 15 is H-NMR spectrum of low and medium molecular weightisoflavone-β-D-glucan according to the present invention;

FIG. 16 is UV spectrum of low and medium molecular weightisoflavone-β-D-glucan according to the present invention;

FIG. 17 shows the composition of monosaccharide of low and mediummolecular weight isoflavone-β-D-glucan according to the presentinvention;

FIG. 18 shows the cytotoxic effects of the products of 80 EP treatedwith autolysis enzyme on human colon cancer Caco-2 cells;

FIG. 19 shows the comparison of cytotoxicity of 80 EP treated with tube#8 with various concentrations; and

FIG. 20 shows the effect of 80 EP treated with autolysis enzyme on thereduction of ICR female mouse body weight induced by lipopolysaccharide(LPS).

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in detail, inconjunction with various examples. These examples are provided only forillustrative purposes, and the present invention is not to be construedas being limited to these examples.

First of all, a mushroom strain having highest activity of autolysisenzyme was selected. In an experiment for determining the activity ofautolysis enzyme by measuring the change of viscosity in the liquidculture medium of Agaricus blazei (AB), Pleurotus ostreatus (PO),Coprinus comatus (CC), Lentinus edodes (LE), Phellinus linteus (PL) andGanoderma lucidum (GL), Agaricus blazei (AB) was confirmed to have thehighest activity.

FIG. 1 shows a process of preparing a low and medium molecular weightisoflavone-β-D-glucan according to the present invention. As shown inFIG. 1, the process comprises step (1) of producing high molecularweight isoflavone-β-D-glucan by culturing AB mycelia in a liquid mediumcontaining isoflavone.

In the next step (2), the high molecular weight isoflavone-β-D-glucan isseparated from the liquid culture of AB mycelia by extracting the liquidculture with boiling water, concentrating the extract, adding ethanol tothe concentrated extract to make precipitation and separating theprecipitate. In the process of treating the extract with ethanol, 80%ethanol has the maximum effect on obtaining high molecular weightisoflavone-β-D-glucan.

Further, the process of preparing a low and medium molecular weightisoflavone-β-D-glucan according to the present invention comprises step(3) of separating autolysis enzyme from a separate liquid culture of ABmycelia.

The next process of preparing isoflavone-β-D-glucan is step (4) ofadding the autolysis enzyme obtained from the liquid culture of ABmycelia to the high molecular weight isoflavone-β-D-glucan to produce alow and medium molecular weight isoflavone-β-D-glucan.

The autolysis enzyme has the best activity in the condition of pH 4.5 to5.5 and at the temperature of 53° C. for 1 to 3 hours. Considering theviscosity of product, the reaction for 3 hours appeared to be optimum.However, the effect of reaction time on producing low and mediummolecular weight isoflavone-β-D-glucan had no difference between 3 hoursand 1 hour. Accordingly, the reaction is preferable to be carried outfor 1 hour.

The process of preparing isoflavone-β-D-glucan according to the presentinvention further comprises step (5) of separating and purifying the lowand medium molecular weight isoflavone-β-D-glucan. The low and mediummolecular weight isoflavone-β-D-glucan is separated by DEAE columnchromatography. The separated fraction is concentrated by vacuumconcentrator and then separated by silica gel column chromatography.

FIG. 12 shows another process of preparing a low and medium molecularweight isoflavone-β-D-glucan according to the present invention. In FIG.12, steps 6 and 8 are carried out by the same procedure of steps 1 and 5in FIG. 1. However, step 7 in FIG. 12 are carried out by adjusting thepH of culture medium to 4.5-5.5 for 1-3 hours instead of treating thehigh molecular weight isoflavone-β-D-glucan with separated autolysisenzyme.

PREPARATIVE EXAMPLE Selection of Mushroom Strain for the Production ofAutolysis Enzyme

Agaricus blazei (AB), Pleurotus ostreatus (PO), Coprinus comatus (CC),Lentinus edodes (LE), Phellinus linteus (PL) and Ganoderma lucidum (GL)were cultured in liquid culture medium at 25° C. for three days in thecondition of 1 v/v/m aeration. Liquid culture of each strain (10 ml) andpolysaccharides prepared from the liquid culture were added with 80%ethanol (80 EP). The mixtures were then incubated at 53° C. and 63° C.for five hours. Degree of autolysis was determined by measuring thechange of viscosity of 80 EP before and after the reaction. Thefollowing Table 1 shows the change of viscosity. TABLE 1 MushroomViscosity (ml/sec) strains 0 hr After 5 hrs Δvis/hr AB 18,850 1,5003,470 PO 1,250 650 120 CC 1,310 790 104 LE 1,120 970 30 PL 1,570 1,10090 GL 1,750 1,210 108

As shown in Table 1, AB has the highest value of 3,470 ml/sec in thechange of viscosity (Δviscosity/hour). PO and CC also have rather highervalues of 120 and 104 ml/sec. As a result, it is considered that AB hasthe highest degree of autolysis compared to other strains.

Example 1 Production and Separation of High Molecular WeightIsoflavone-β-D-glucan

The culture medium contained soybean powder or natural source includingsoybean powder. Natural isoflavone separated from soybean or syntheticisoflavone may also be used. The liquid medium were added with brownsugar as carbon source and inorganic salts, and then autoclaved at 120°C. for 30 minutes.

AB mycelia were inoculated in the liquid culture medium containingisoflavone and cultured with aeration or stirring for 1 to 7 days toproduce high molecular weight isoflavone-β-D-glucan. The molecularweight of the high molecular weight isoflavone-β-D-glucan was at least30,000.

Liquid culture of AB mycelia was extracted with boiling water at 100° C.for 10 hours, and the extract was autoclaved at 121° C. for 1 hour.

The extract of liquid culture of AB mycelia was treated with 80% ethanolto make precipitation and then centrifuged to remove supernatant. Theprecipitate was separated to obtain high molecular weight isoflavonewith β-D-glucan having the molecular weight of at least 100,000.

Example 2 Selection of Ethanol Fraction Having the Highest Content ofHigh Molecular Weight Isoflavone-β-D-glucan

The extract of liquid culture of AB mycelia was suspended in ethanol toprepare 10 to 80% ethanol solution to induce precipitation. Eachprecipitate was separated and then applied to Biosep S-2000 column.

FIG. 2 is HPLC chromatograms of the extract of liquid culture ofAgaricus blazei mycelia treated with various concentrations of ethanolsolution (40, 50, 60, 70 and 80% ethanol) and then separated with BiosepS-2000 column. The chromatogram on the right side below represents thechromatogram of total extract of liquid culture of AB mycelia. In FIG.2, horizontal axis represents time (minutes) and vertical axisrepresents length (mAU).

As shown in FIG. 2, the high molecular weight isoflavone-β-D-glucan,obtained by treating 80% ethanol to the liquid culture of AB mycelia toinduce precipitation, hereinafter referred to ‘80 EP (80% ethanolprecipitate)’, shows similar chromatograph pattern to 70 EP, bothshowing peaks at RT (retention time) 2.2 and 3. In 80 EP, the peak at RT3 is higher than the peak at RT 2.2, while two peaks at RT 3 and 2.2have similar heights to those of 70 EP. Other fractions have similarpatterns to the total extract of liquid culture of AB mycelia, showingpeaks at RT 2.2, 3 and 3.6.

From the above, it is considered that 80 EP has higher amount of highmolecular weight isoflavone-β-D-glucan than other ethanol precipitateshave. Further, 80 EP does not have unnecessary peaks, such as the peakat RT 3.6 which is found in other EP. Accordingly, 80 EP is used toincrease the yield of isoflavone-β-D-glucan according to the presentinvention.

Example 3 Separation and Fractionation of Autolysis Enzyme

The separation of autolysis enzyme was carried out by filtrating theliquid culture of AB mycelia at reduced pressure. TCA (trichloroaceticacid) was added to the filtrate to the concentration of 10% and themixture was placed at 4° C. for 24 hours. The resultant was centrifugedat 4° C. and 10,000 rpm for 15 minutes to obtain the precipitate.

The precipitate was further fractionated by DEAE (diethylaminoethyl)column chromatography and each fraction was determined its UV absorbanceat 280 nm.

FIG. 3 shows fractionation of the precipitate of the liquid culture ofAgaricus blazei mycelia by DEAE column chromatography. In FIG. 3,horizontal axis represents number of each fraction and vertical axisrepresents UV absorbance at 280 nm. As shown in FIG. 3, UV absorbance ofeach fraction was monitored and eight fractions were obtained showing UVabsorbance at 280 nm. Fraction #2 (tube #8) showed the maximum UVabsorbance. When autolysis activity of each fraction was tested, tube #8showed the highest activity. Accordingly, tube #8 was used for treatingthe 80 EP obtained in Example 2 to prepare low and medium molecularweight isoflavone-β-D-glucan.

FIG. 4 is UV spectrum of DEAE column fraction #2 (tube #8) at 225-445nm. In FIG. 4, horizontal axis represents wavelength, vertical axisrepresents absorbance, and the left box shows the numerical value ofwavelength and absorbance. As shown in FIG. 4, fraction #2 (tube #8) hasthe maximum UV absorbance at 270 nm.

Example 4 Determination of Autolysis Enzyme Activity

The fraction of DEAE column chromatography was added to 80 EP obtainedin Example 2. The production of low and medium molecular weightisoflavone-β-D-glucan from high molecular weight isoflavone-β-D-glucanwas confirmed by using TSK column and C18 column. In TSK columnchromatography, H₂O was used as mobile phase at 1 ml/min. In C18 columnchromatography, MeOH:1 mM ammonium acetate (6:4) was used as mobilephase at 1 min. Isoflavone was identified at UV 257 nm and 267 nm. RIdetector was used to identify β-D-glucan.

Example 5 Determination of Molecular Weight of Autolysis Enzyme

As tube #8 obtained in Example 3 showed maximum autolysis activity,molecular weight of the protein contained in tube #8 was determined bySDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis).

The fraction of tube #8 was dried and then SDS-PAGE sample buffer wasadded to dissolve the resultant. Electrophoresis was performed on 12%SDS-PAGE according to Laemmli. Size markers used were β-galactosidase(175 kDa), paramyosin (83 kDa), glutamic dehydrogenase (62 kDa),aldolase (48 kDa), triosephosphate isomerase (33 kDa), β-lactoglobulin A(25 kDa), lysozyme (17 kDa) and aprotinin (7 kDa)

FIG. 5 shows SDS-PAGE pattern of tubes #8 and #14. In FIG. 5, SM ofhorizontal axis is size marker and vertical axis represents molecularweight of the protein used as size marker. As shown in FIG. 5, molecularweight of the protein in tube #8 corresponds to 7 kDa, which shows thatmolecular weight of autolysis enzyme is about 7 kDa.

Example 6 Production of Low and Medium Molecular WeightIsoflavone-β-D-glucan by autolysis enzyme

After adjusting its pH to 5.5, the high molecular weightisoflavone-β-D-glucan obtained by treating 80% ethanol to the liquidculture of AB mycelia (80 EP) was added with 10 ml of autolysis enzymeseparated from DEAE column in Example 3. The mixture was placed at 53°C. for 1 hours to produce a low and medium molecular weightisoflavone-β-D-glucan. The molecular weight of obtainedisoflavone-β-D-glucan was less than about 30,000.

In order to determine the optimum condition for the activity ofautolysis enzyme obtained from the liquid culture of AB mycelia, theliquid culture of AB mycelia and 80 EP were reacted at each conditionsof pH 4.5, 5.5, 6.5 and 7.5 and temperature of 53° C. and 63° C. for 1,3, 5, 15 and 24 hours and the viscosity was measured in each condition.

Table 2 shows the viscosity of liquid culture of AB mycelia reacted withautolysis enzyme. As shown in Table 2, the autolysis enzyme obtainedfrom AB mycelia has maximum activity at the condition of pH 4.5 to 5.5and the temperature of 53° C. for 3 hours of reaction. TABLE 2 53° C.63° C. Reaction time pH 4.5 pH 5.5 pH 6.5 pH 7.5 pH 4.5 pH 5.5 pH 6.5 pH7.5 0 18,750 18,850 19,300 19,750 18,750 18,850 19,300 19,750 1 hr16,250 16,950 17,700 16,500 17,250 15,750 18,250 16,650 3 hrs 1,5001,500 12,500 2,300 7,750 15,500 16,000 16,500 5 hrs 1,500 1,650 6,0002,050 1,550 3,650 4,300 4,200 15 hrs 1,650 1,500 1,650 1,550 1,550 1,5001,900 1,700 24 hrs 1,650 1,650 1,500 1,600 1,500 1,750 1,750 1,750

As shown in Table 2, the viscosity of liquid culture of AB myceliadecreased from 18,750 to 1,500 after the reaction for 3 hours at thecondition of pH 4.5 and 53° C. As a result, it is found that theautolysis enzyme has maximum activity at the condition of pH 4.5 to 5.5and the temperature of 53° C. for 3 hours of reaction

Example 7 Identification of Low and Medium Molecular WeightIsoflavone-β-D-glucan

Low and medium molecular weight isoflavone-β-D-glucan was identified byTLC and HPLC.

(1) Identification by TLC (Thin-Layer Chromatography)

Free sugars or polymeric sugar has no UV absorbance. Therefore, in orderto examine that an area separated by TLC has the material having UVabsorbance, sugars, or sugars having UV absorbance, the separated bandwas detected with UV lamp and also analyzed by color development withdiphenylamine aridine phosphate (DAP).

The fraction separated by silica gel column chromatography was developedby TLC (Silica gel 60F-254 plate, 5×10 cm). The mobile phase wasbutanol:ethanol:H₂O (5:3:3) (v/v/v). The separated band was treated byDAP and UV lamp was used to identify the material having UV absorbance.

FIG. 6 shows TLC pattern of 80 EP reacted with autolysis enzyme. Inhorizontal axis of FIG. 6, DP7 represents maltoheptose; DP4,maltotetrose; DP3, maltotriose; DT 5,000, dextran 5,000; DT 12,000,dextran 12000; DT 25,000, dextran 25,000; P+80% ethanol, 80 EP treatedwith autolysis enzyme; and 80% ethanol, 80 EP without treatment withautolysis enzyme. The vertical axis of FIG. 6 represents Rf value whichmeans the separation of material.

As shown in FIG. 6, low molecular weight polysaccharides (DP7 or less)move, while medium or higher molecular weight polysaccharides (MW 5,000)do not moved. In high molecular weight polysaccharides treated withautolysis enzyme, low molecular weight sugars (DP3 or less) showingcolor development by DAP are mixed with the materials having UVabsorbance and showing color development by DAP. Especially, medium orhigher molecular weight polysaccharides showing no movement have UVabsorbance, and sugars of DP7 also have UV absorbance. Further, thematerials having UV absorbance only are also included herein.

The above results show that high molecular weight polysaccharides areconverted to medium and low molecular weight materials by autolysis andthat the medium and low molecular weight materials are combined with thematerials having UC absorbance.

(2) Separation by HPLC (High Performance Liquid Chromatography)

Bio-Sep S2000 column (mobile phase; 20 mM sodium phosphate), TSK column(mobile phase; H₂O) and C18 column (mobile phase; MeOH:1 mM ammoniumacetate (6:4)) were used. Flow rate was 1 ml/min in all cases. UVdetector (UV 257 nm, 267 nm, 280 nm) and RI detector was used.

FIGS. 7 a and 7 b are HPLC chromatograms of each fraction containingautolysis enzyme separated from HPLC (TSK column) before and aftertreating with 80 EP. The horizontal axis represents time (minutes) andthe vertical axis represents length (mAU). The left panel shows HPLCchromatograms of each fraction of #2, #8, #14, #25, #35, #65 and #83treated with 80 EP a shows HPLC chromatograms of each fraction.

As shown in FIGS. 7 a and 7 b, the uppermost chromatogram of 80 EP hasfour peaks. When each fraction of autolysis enzyme separated from DEAEchromatography (FIG. 3) was reacted with 80 EP, the fraction of tube #8showed different pattern from that of 80 EP, which represents that newproduct has been formed. In the other fractions, however, the originalpattern of 80 EP was maintained, which means that digestion has not beencarried out. Accordingly, it is considered that the high molecularpolysaccharides contained in 80 EP have been digested into medium or lowmolecular weight polysaccharides by the reaction with the fraction oftube #8.

Subsequently, the digest obtained from 80 EP treated with autolysisenzyme (tube #8) was separated with HPLC, and then monitored by UVdetector and RI detector simultaneously.

FIG. 8 are HPLC chromatograms of the products from 80 EP treated withtube #8, which are detected with UV detector and RI detector. In FIG. 8,left panel A shows the result of RI detector and right panel B shows theresult of UV detector (267 nm). As shown in FIG. 8, only RT 8.8 peak isdetected by both UV detector and RI detector, and the other peaks aredetected either UV detector or RI detector. Considering the pattern ofRT 8.8 peak, it is confirmed that polysaccharides are coupled withanother material (having UV absorbance) which is considered to beisoflavone having UV absorbance.

Example 8 Confirmation of Production of Low and Medium Molecular WeightIsoflavone-β-D-Glucan by Autolysis Enzyme

In order to confirm that low and medium molecular weightisoflavone-β-D-glucan was produced by autolysis enzyme during the liquidculture of AB mycelia, TLC was carried out.

When soybean powder (SP) was fractionated by ethanol (10, 20, 30, 40,50, 60, 70 and 80%) and then each precipitate was separated, the mostamount of precipitate was obtained in 70% ethanol, little amount wasobtained in 10 to 60% ethanol, and small amount was obtained in 80%ethanol. Considering the most amount of precipitate derived from theliquid culture of AB mycelia has been obtained in 80 EP, it isconsidered that the precipitate contains such material that is notcontained in SP but produced during the liquid culture of AB mycelia bybioconversion.

FIG. 9 shows TLC pattern of soybean powder (SP) and its acid hydrolyzate(SPH). In FIG. 9, AP means precipitate of 80 EP obtained from the liquidculture of AB mycelia and then treated again with 80% ethanol; APH,acid-hydrolyzate of AP; AS, supernatant of the above 80 EP; ASH,hydrolyzate of AS; SP, soybean powder consisting of culture media; SPH,hydrolyzate of SP. G (genistein), D (daidzein) and GT (genisteinstandard) are markers and the vertical axis represents Rf valuesthereof.

As shown in FIG. 9, all the samples obtained from SP contain thematerials having UV absorbance (RF 0.999, 0.542, 0.306) alone(especially, G and D, etc.) and do not contain the materials detected inUV lamp and DAP simultaneously, which shows that SP provides thematerials having UV absorbance alone. On the other hand, the fractionobtained from AP has the material detected in UV and DAP simultaneously(Rf 0.542, 0.014), which means that the material is not contained inculture medium but produced during the liquid culture of AB mycelia bybioconversion.

FIG. 10 is UV/VIS spectra of autolysis enzyme-treated 80 EP and FIGS. 11a, 11 b and 11 c are IR spectra of TLC bands of FIG. 9. As shown inFIGS. 10, 11 a, 11 b and 11 c, all the spectra are similar to those of Gand D, which means that they are glycosides of G or D. It is consideredthat the materials are produced during the liquid culture bybioconversion.

According to the above Examples 7 and 8, it is confirmed that highmolecular weight β-D-glucan is digested to low and medium molecularweight β-D-glucan by autolysis enzyme, and the product is not madeexclusively of low and medium molecular weight β-D-glucan but formed incombination with a certain material which is not polysaccharide.

Example 9 Identification of Isoflavone Contained in Final Product

Isoflavone-β-D-glucan which is finally separated and purified accordingto the present invention (A) and isoflavone-β-D-glucan hydrolyzed byenzyme (β-glucosidase and megazyme kit) (B) were analyzed by HPLC (C18column, mobile phase; MeOH: 1 mM ammonium acetate (6:4), flow rate; 1ml/min).

Table 3 shows the content of free genistein and daidzein in A and B.TABLE 3 Genistein Daidzein isoflavone-β-D- — — glucan(A)isoflavone-β-D-glucan 150 mg/g dry weight 28.2 mg/g dry weighthydrolyzate (B)

As shown in Table 3, no free genistein and daidzein was detected in Aand 150 mg/g dry weight of genistein and 28.2 mg/g dry weight ofdaidzein were detected in B. The result means that the final product ofthe present invention does not contain genistein and daidzein, free formof isoflavone, while the hydrolyzate of the final product contains them.Accordingly, it is considered that isoflavone is contained in the finalproduct of the present invention (A), which contains isoflavone andβ-D-glucan not as mixture but as glycoside.

Example 10 Determination of Molecular Weight of Low and Medium MolecularWeight Isoflavone-β-D-glucan

Molecular weight of low and medium molecular weightisoflavone-β-D-glucan was determined by HPLC equipped with TSK column(PDA-100 UV detector and RI detector, P-680 pump, ASI-100 fractioncollector, Dionex). Mobile phase was triple distilled water and flowrate was 1 ml/min.

FIG. 13 is a graph showing molecular weight of low and medium molecularweight isoflavone-β-D-glucan according to the present inventioncalculated by comparing with dextran standard and displayed in logscale. As shown in FIG. 13, molecular weight of low and medium molecularweight isoflavone-β-D-glucan was about 25,000.

Example 11 Structure of Low and Medium Molecular WeightIsoflavone-β-D-glucan

Low and medium molecular weight isoflavone-β-D-glucan was separatedagain by HPLC (TSK column, mobile phase; H₂O) and then a fractionshowing the greatest anti-cancer effect was collected. In the fraction,sugar and isoflavone (genistein, daidzein) was determined by IR, H-NMRand UV spectra.

FIG. 14 is IR spectrum of low and medium molecular weightisoflavone-β-D-glucan according to the present invention. In IRspectrum, the characteristic peaks were 495-656 cm⁻¹ (strong, broad),1014 cm⁻¹ (strong, narrow), 1109 cm⁻¹ (weak, narrow), 1402.0, 1450.8,1472.7 cm⁻¹ (weak, broad), 1642.6 cm⁻¹ (strong, narrow), 2097.0-2112.0cm⁻¹ (medium weak, broad), 2390 cm⁻¹ (very weak, broad), 2843 cm⁻¹(medium, narrow in OH absorbance ˜3000 cm⁻¹), 3173.0-3648 cm⁻¹ (broad inOH absorbance ˜3000 cm⁻¹), the characteristic peaks shown in IR spectrumof G or D standard.

FIG. 15 is H-NMR spectrum and FIG. 16 is UV spectrum of low and mediummolecular weight isoflavone-β-D-glucan according to the presentinvention. As shown in FIG. 15, maximum absorbance was appeared in 267nm, likewise that of G. Accordingly, it is considered that RT 8.8 peakindicates β-D-glucan containing mostly G among isoflavone.

Example 12 Composition of Sugars

Sample was added with 5 ml of phosphate buffer (20 mM, pH 6.5) and thendigested by megazyme kit (β-glucosidase; 0.2 U, 0.1 ml) and licenase (10U, 0.2 ml). The digest was analyzed by HPLC (Rezo RCM-monosaccharidecolumn, 200×10 mm) to identify the composition of sugars.

FIG. 17 shows the composition of monosaccharide of low and mediummolecular weight isoflavone-β-D-glucan according to the presentinvention (RT 8.8). As shown in FIG. 17, the low and medium molecularweight isoflavone-β-D-glucan according to the present invention (RT 8.8)includes β-D-glucose, D-fructose and ribose.

Preparation Example 1 Production of High Molecular WeightIsoflavone-β-D-glucan (MW 30,000 or more) by Culturing AB Mycelia inLiquid Medium Containing Isoflavone

40 g of soybean or soybean powder was added with 10 g of protease and 10g of cellulase and then digested at 60° C. and 200 rpm for 1 to 5 hours.The digest may be used with or without addition of 10 to 1000 mg ofnatural isoflavone separated from vegetables or synthetic isoflavone.220 g of brown sugar, 3 g of ribose and inorganic salts (10 g of MgSO₄,10 g of KH₂PO₄) were added thereto and then water was added to make thetotal volume 1 l. The mixture was autoclaved at 121° C. for 1 hour tomake liquid culture medium.

Agaricus blazei mycelia were inoculated into the liquid medium, and thencultured upon shaking or aeration at 25-35° C. for 1-7 days.

Preparation Example 2 Separation of High Molecular WeightIsoflavone-β-D-glucan

The culture obtained in Preparation Example 1 was autoclaved at 121° C.for 1 hour, filtrated through diatomite, and then concentrated to 1/10volume under vacuum. 200 g of the concentrate was added with ethanol tobe 80% ethanol solution and then mixed. The mixture was placed at 4° C.for 24 hours to induce precipitation. The precipitated liquid wascentrifuged at 10,000 rpm for 10 minutes and the supernatant was removedto obtain 3.8 g of the precipitate containing β-D-glucan having themolecular weight of 100,000 or more (80 EP).

Preparation Example 3 Separation and Purification of Autolysis EnzymeFrom Liquid Culture of Agaricus blazei Mycelia

Liquid culture of AB mycelia was filtrated under reduced pressure andthe filtrate was mixed with TCA to make 10% TCA solution. The mixturewas placed at 4° C. for 24 hours and then centrifuged at 4° C. and10,000 rpm for 15 minutes to separate the precipitate.

The precipitate was dissolved in 50 mM sodium acetate buffer (pH 5.0)and then separated by DEAE column (2 cm, 110 cm) to collect factions by10 ml. The mobile phase was 20 mM sodium phosphate buffer (20 mM NaH₂PO₄and 20 mM Na₂HPO₄, pH 7.0).

Preparation Example 4 Production of Low and Medium Molecular WeightIsoflavone-β-D-glucan

After adjusting the pH to 5.5, 80 EP obtained in Preparation Example 2was added with 10 ml of autolysis enzyme separated from DEAE column, andthen the mixture was incubated in shaking incubator at 53° C. for 1hour.

The production of low and medium molecular weight isoflavone-β-D-glucan(MW 30,000 or less) by autolysis enzyme was confirmed by TLC and HPLC asmentioned above.

Preparation Example 5 Separation and Purification of Low and MediumMolecular Weight Isoflavone-β-D-glucan

(1) DEAE Column Chromatography

3 ml of the autolyzed product obtained in Preparation Example 4 wasloaded on DEAE column (2 cm, 110 cm) filled with 5 mM sodium phosphatebuffer (pH 7.7) and every 310 drops (10 ml) fractions were collected.The mobile phase was 5 mM sodium phosphate buffer (5 mM NaH₂PO₄ and 5 mMNa₂HPO₄, pH 7.7).

(2) Silica Gel Column Chromatography

The fraction obtained by DEAE column chromatography was concentrated byvacuum concentrator and separated again by silica gel columnchromatography. The mobile phase was butanol:ethanol:H₂O (5:3:3).

Low and medium molecular weight isoflavone-β-D-glucan in the separatedfraction was confirmed by TLC and HPLC as mentioned above.

Hereinafter, the anti-cancer effect and immunoenhancing effect of lowand medium molecular weight isoflavone-β-D-glucan as produced above willbe described in detail, in conjunction with various examples.

Experiment 1: Cytotoxicity Against S-180 Ascites Cancer Cells

S-180 cells were maintained and subcultured at 37° C. in a humidifiedCO₂ incubator (95% air-5% CO₂). The complete medium for cell maintenanceconsisted of DMEM (Dulbecco's modified Eagle's medium) containing 10%horse serum, 100 U/ml penicillin and 100 μl/ml streptomycin. When cellswere 80% confluent, the medium was changed every two days.

10 mg/ml of sample in double distilled water (DDW) was diluted by 10fold in DMEM to make a sample solution containing 100 μl of DDW and 900μl of DMEM, while control solution contained 100 μl of DDW and 900 μl ofDMEM. 1.5×10⁵ cells/ml of S-180 cells were diluted with DMEM to be 5×10⁴cells/ml of DMEM.

To measure the effect of 80 EP on cell growth curves, cells were platedin complete medium in 24-well plates. All wells were treated with 5×10⁴cells/ml DMEM and then with each sample or control. The treated wellplates were cultured in 5% CO₂ incubator at 37° C. for 48 hours. Alivecells were dyed by 0.2% tryphane blue and the number of viable cells wasdetermined by counting in a hemocytometer to calculate ED₅₀.

Table 4 shows the toxicity of the sample from 80 EP fraction treatedwith autolysis enzyme (tube #8) against S-180 cancer cells. As shown inTable 4, the cell number of control was 23.7×10⁴ cells/ml after 48 hoursof incubation. The cell numbers treated with autolyzed AB in eachconcentration of 10, 20 and 30 μg/ml were 7.5×10⁴, 6.0×10⁴ and 5.5×10⁴cells/ml, respectively, resulting in 0.9 μg/ml of ED₅₀. On the otherhand, the cell numbers treated with AB without autolysis in eachconcentration of 10, 20 and 30 μg/ml were 11.0×10⁴, 9.5×10⁴ and 8.5×10⁴cells/ml, respectively, resulting in 2.1 μg/ml of ED₅₀. The result showsthat the cytotoxicity increases in the sample treated with autolysisenzyme. Accordingly, it is considered that cytotoxic substances aregenerated from 80 EP fraction by the treatment of autolysis enzyme.TABLE 4 Doses Growth of Cells Growth Ratio ED₅₀ Treatment (μg/ml) (×10⁴cells/ml) (%) (μg/ml) Control 10 20 23.7 ± 2.5  100 — 30 AB 10 11.0 ±0.7  31.11 20 9.5 ± 1.4 23.32 2.1 30 8.5 ± 0.7 18.14 Autolyzed 10 7.5 ±0.7 12.95 AB 20 6.0 ± 0.1 5.18 0.9 30 5.5 ± 1.4 2.59

Experiment 2: Anticarcinogenicity Against Mouse Ascite Cancer

7 week-old female ICR (Institute for Cancer Research) mice were obtainedfrom Life Science (Taegu, Korea). S-180 cells were supplied by KoreanCell Line Bank (Seoul, Korea). The culture medium for S-180 was obtainedby GIBCO and other reagents used were first grade.

The ICR mice were grouped by 10 mice so that each group had equal meanbody weight and then each group was put into a cage. The mice wereraised freely with food and water for 1 week in an animal house atcontrolled room temperature and relative humidity. S-180 cells weresubcultured as ascites type in ICR mice, and the tumor cells wereharvested from the abdominal cavities of the mice 7 days after theimplantation. 0.1 ml of S-1 80 cells (1×10⁷ cells/ml PBS) wastransplanted subcutaneously into the abdomen of each mice to induceascite cancer. 0.1 ml of test sample, dissolved or suspended in PBS(0.01M, pH 7.0) in adequate concentrations, was injectedintraperitoneally every two days for 2 weeks, starting 24 hours aftertumor implantation. After the intraperitoneal injection of S-180, theweight of mouse and the amount of feed intake were measured every threedays for 40 days. The number of survived mice and the number of survivaldays were also measured.

Table 5 shows the number of survived mice and survival days of controlgroup, the group treated with 80 EP, and the group treated withautolyzed 80 EP. As shown in Table 5, the average survival days ofcontrol group was 19.2 days, while it was extended to 26.9 days(lengthen the life span by 40%) by the treatment of autolyzed AB and 3mice were still alive after 40 days. The treatment of AB also lengthenedsurvival days compared with control but less effective than autolyzedAB. TABLE 5 Treatment Mean survival days Survival rate (%) Survived miceControl 19.2 100 0/10 AB 22.3 116 0/10 Autolyzed AB 26.9 140 3/10

Experiment 3: Cytotoxicity Against Human Cancer Cell Lines

(1) Medium: 1 pack of DMEM was dissolved in 900 ml of triple distilledwater and then added with 1 ml of penicillin-streptomycin. The mixturewas added with 2 g of sodium bicarbonate and 100 ml of FBS (fetal bovineserum), and then filtrated by filter paper (0.22 μm). The completemedium for cell maintenance was stored at 4° C.

(2) Cell culture: Human cancer cell lines (MCF-7, breast cancer andHela, uterine cervix cancer cell) were subcultured in DMEM medium at 37°C. for 24 hours in 5% CO₂ incubator. The cultured cell lines weretreated with 1 ml of trypsin-EDTA at 37° C. for 10 minutes so that cellswere separated from medium. Cells were recovered by centrifugation(1,500 rpm, 2 minutes) and then dissolved in the above medium. 1 ml ofthe medium containing cells was distributed in 12 well-plates so thateach well contained 5×10⁴ cells.

(3) Cytotoxicity Against Hela Cell and MCF-7 Cell

Table 6 shows the cytotoxicity of the samples from 80 EP fractiontreated with or without autolysis enzyme (tube #8) against Hela cell(uterine cervix cancer cell line). As shown in Table 6, the cell numberof Hela cells was 20.1×10⁴ cells/ml after 48 hours incubation. ED₅₀values of AB and autolyzed AB were 5.6 and 0.9 μg/ml, respectively.Accordingly, it is considered that autolyzed AB has strong cytotoxicityand cytotoxic substances are produced from 80 EP fraction by thetreatment of autolysis enzyme. TABLE 6 Doses Growth of Cells GrowthRatio ED₅₀ Treatment (μg/ml) (×10⁴ cells/ml) (%) (μg/ml) Control 10 2020.1 ± 1.1  100 — 30 AB 10 11.0 ± 1.4  36.4 20 9.5 ± 1.4 27.2 5.6 30 7.0± 0.7 12.1 Autolyzed 10 7.0 ± 0.7 18.1 AB 20 6.0 ± 0.1 15.1 0.9 30 5.5 ±1.4 3.0

Table 7 shows the cytotoxicity of the samples from 80 EP fractiontreated with or without autolysis enzyme (tube #8) against MCF-7 cell(breast cancer cell line). As shown in Table 7, the cell number of MCF-7cells was 21.5×10⁴ cells/ml after 48 hours incubation. ED₅₀ values of ABand autolyzed AB were 5.8 and 1.2 μg/ml, respectively, which weresimilar to those for Hela cells. TABLE 7 Doses Growth of Cells GrowthRatio ED₅₀ Treatment (μg/ml) (×10⁴ cells/ml) (%) (μg/ml) Control 10 2021.5 ± 2.1  100 — 30 AB 10 12.0 ± 1.4  39.3 20 9.5 ± 1.4 27.2 5.8 30  80± 1.4 18.1 Autolyzed 10 8.5 ± 0.7 21.2 AB 20 7.0 ± 0.1 18.1 1.2 30 5.5 ±1.4 6.1

(4) Cytotoxicity Against Caxo-2 Cells

80 EP fraction treated with autolysis enzyme (tube #8 or fractionated byHPLC as FIG. 5) was fractionated by DEAE cellulose column chromatographyto collect 6 fractions (tube #4, #14, #25, #32, #68 and #83).

FIG. 18 shows the cytotoxic effects of the products of 80 EP treatedwith autolysis enzyme on human colon cancer Caco-2 cells. In FIG. 18,the height of bar represents percentage of cell number after thetreatment of autolysis enzyme as compared with control. As shown in FIG.18, 80 EP fraction without treatment of autolysis enzyme shows 85.7% ofviability, that is, 14% of cytotoxicity. On the other hand, tube #4 andtube #14 treated with autolysis enzyme show 37% and 33% of cytotoxicity,respectively. Other samples also show toxicity at a lesser extent. Theseresult also suggest that cytotoxic substances are produced from 80 EPfraction by the treatment of autolysis enzyme.

FIG. 19 shows the comparison of cytotoxicity of 80 EP treated with tube#8 with various concentrations. In FIG. 19, sample A is 80 EP withouttreatment of autolysis enzyme and sample B is 80 EP treated withautolysis enzyme. The height of bar represents percentage of cell numberafter the treatment of autolysis enzyme as compared with control, inwhich concentration of white bar, dotted bar and shaded bar are 1, 5 and10 mg, respectively. In sample A, cytotoxicity was not affected by theincrease of concentration, while in sample B, cytotoxicity increased to2, 23 and 42%, respectively, with the treatment of autolysis enzyme by1, 5 and 10 mg. These result also suggests that cytotoxic substances areproduced from 80 EP fraction by the treatment of autolysis enzyme.

Experiment 4: Immunoenhancing Effect of Isoflavone-β-D-glucan

Female ICR mice (6-7 weeks of age) were housed in polycarbonated cage (4mice/cage) and raised with feeding pellet fodder for mouse for 1 weeksand those of 28±1 g body weight were selected for experiment. All micewere raised freely with food and water in an animal house at 22±1° C.room temperature and 50% relative humidity maintaining light-and-darkcycle of 12 hour interval.

One week later, the mice were subjected to one of the followingtreatments for 14 days: 0 (control), 1, 2 and 4 mg/g body weight/0.2 mldistilled water. Samples were injected per os. At the end of 14consecutive feeding days, mice were weighted and injectedintraperitoneally with 1 mg/kg body weight of lipopolysaccharide (LPS,Sigma Chemical Co.) in sterile HEPES buffer (Sigma Chemical Co.) orHEPES buffer alone. All mice were weighted 4, 8, 12, 24, 48 and 72 hourspost injection.

FIG. 20 shows the effect of 80 EP treated with autolysis enzyme on thereduction of ICR female mouse body weight induced by lipopolysaccharide(LPD). In FIG. 20, (1) represents control group; (2), (3) and (4)represent the groups treated with 1, 2 and 4 mg of autolyzed 80 EP,respectively; and (5), (6) and (7) represent the groups treated with 1,2 and 4 mg of 80 EP without treatment of autolysis enzyme.

LPS, a complex of lipid and polysaccharide with a covalent bond, formsan endotoxin consisting in outer membrane component of Gram negativebacteria. LPS has various biological activity and is called endotoxin.LPS of bacteria is recognized as the signal of infection by the immunesystem of host. Accordingly, immune ability may be presumed bydetermining the reduction of body weight after the treatment of LPS.

As shown in FIG. 20, all mice treated with LPS showed reduction of bodyweight. In control mice treated with LPS, body weight decreaseddramatically with the lapse of time and resulted in 2.42 g reduction in24 hours after the treatment of LPS. The treatment of 80 EP treated withautolysis enzyme (autolyzed AB) suppressed the reduction of body weightas compared with that of control. In autolyzed AB groups, the reductionsof body weight in 24 hours after the treatment of LPS were −1.77, −1.22and −0.75 g, respectively, with the treatment of 1, 2 and 4 mg ofautolyzed AB. Accordingly, it is considered that autolyzed AB may reducethe effect of LPS. The AB sample (80 EP without treatment of autolyzedenzyme) was less effective for the suppression of body weight reduction,compared to autolyzed AB sample. These results suggest that activesubstances which may suppress the effect of LPS in ICR mice are producedfrom 80 EP fraction by the treatment of autolysis enzyme, which meanslow and medium molecular weight isoflavone-β-D-glucan hasimmunoenhancing effect.

INDUSTRIAL APPLICABILITY

The present invention provides low and medium molecular weightisoflavone-β-D-glucan produced by submerged liquid culture of Agaricusblazei, a method of producing the isoflavone-β-D-glucan using autolysisenzyme of Agaricus blazei mycelia, and use of the isoflavone-β-D-glucanfor anti-cancer and immunoenhancing effect.

According to the present invention, low and medium molecular weightisoflavone-β-D-glucan is prepared from high molecular weightisoflavone-β-D-glucan extracted from liquid culture of Agaricus blazeimycelia, by using autolysis enzyme secreted by Agaricus blazei myceliaat an optimum condition for the activity of the enzyme. The processrequires a short period of time and enables the product to be obtainedon a large scale at low cost.

Low and medium molecular weight isoflavone-β-D-glucan of the presentinvention has anti-cancer effect as well as immunoenhancing effectwithout toxic effect on normal cells. The anti-cancer effect isremarkably increased with immunoenhancing effect.

1. A method of producing isoflavone-β-D-glucan, which comprises thesteps of: culturing Agaricus blazei mycelia in a liquid mediumcontaining isoflavone to produce a high molecular weightisoflavone-β-D-glucan; activating an autolysis enzyme in the liquidculture of Agaricus blazei mycelia to produce a low and medium molecularweight isoflavone-β-D-glucan; and separating and purifying the low andmedium molecular weight isoflavone-β-D-glucan.
 2. The method accordingto claim 1, wherein the step of activating an autolysis enzyme from theliquid culture of Agaricus blazei mycelia is carried out by adjustingthe pH of culture medium to pH 4.5-5.5 for 1-3 hours.
 3. Theisoflavone-β-D-glucan produced by the method of claim
 1. 4. Animmunoenhancing agent comprising the isoflavone-β-D-glucan of claim 3.